Institute of Marine and Environmental Technology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 701 E. Pratt St, Baltimore, MD, 21202, USA.
Institute of Sports and Exercise Biology, Shaanxi Normal University, Xi'an, 710062, China.
Mar Biotechnol (NY). 2018 Apr;20(2):168-181. doi: 10.1007/s10126-018-9794-8. Epub 2018 Jan 27.
Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.
斑马鱼胚胎慢肌细胞位于表面，肌节组织清晰，为肌肉细胞分化和肌节组装的遗传分析提供了有用的模型系统。为了开发一种快速检测斑马鱼胚胎慢肌中 CRISPR 介导的基因编辑的方法，我们针对在 myomesin-3-RFP (Myom3-RFP) 斑马鱼胚胎中特异性表达的红色荧光蛋白 (RFP) 报告基因。我们证明，微注射 Cas9 蛋白与 RFP-sgRNA 或 Cas9 mRNA 导致注射斑马鱼胚胎中慢肌纤维 RFP 表达的镶嵌模式。为了揭示肌节组织中基因功能，我们针对两个内源性基因，即慢肌球蛋白重链-1 (smyhc1) 和热休克蛋白 90α1 (hsp90α1)，它们在斑马鱼肌肉细胞中特异性表达。我们证明，注射靶向 smyhc1 或 hsp90a1 的 Cas9 蛋白或 sgRNA 导致注射的斑马鱼胚胎中慢肌纤维肌球蛋白粗丝的破坏呈现镶嵌模式。此外，这些有缺陷的肌纤维中的 Myom3-RFP 表达和 M 线定位也被消除。鉴于斑马鱼胚胎慢肌是一个快速的体内系统，用于测试基因组编辑和揭示肌肉细胞分化中的基因功能，我们研究了 Natronobacterium gregoryi Argonaute (NgAgo) 系统的微注射是否会在斑马鱼胚胎中诱导遗传突变和肌肉缺陷。靶向 RFP、Smyhc1 或 Hsp90α1 的单链引导 DNA 与 NgAgo mRNA 一起注射到 Myom3-RFP 斑马鱼胚胎中。分析了注射胚胎中的 Myom3-RFP 表达。结果表明，与 CRISPR/Cas9 系统相比，NgAgo-gDNA 系统的注射不会影响注射胚胎中肌纤维的 Myom3-RFP 表达和肌节组织。序列分析未能检测到靶基因的遗传突变。总之，我们的研究表明，斑马鱼胚胎慢肌是一种快速的体内模型，用于测试基因编辑技术和揭示肌肉细胞分化中的基因功能。
